Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Harunori Yoshikawa; Mark Larance; Dylan J. Harney; Ramasubramanian Sundaramoorthy; Tony Ly; Thomas Owen-Hughes; Angus I. Lamond

doi:10.7554/eLife.36530

Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Harunori Yoshikawa, Mark Larance, Dylan J. Harney, Ramasubramanian Sundaramoorthy, Tony Ly, Thomas Owen-Hughes, Angus I. Lamond (Lead / Corresponding author)

Gene Regulation and Expression

Research output: Contribution to journal › Article › peer-review

23 Citations (Scopus)

165 Downloads (Pure)

Abstract

We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

Original language	English
Article number	e36530
Pages (from-to)	1-26
Number of pages	26
Journal	eLife
Volume	7
Issue number	7
DOIs	https://doi.org/10.7554/eLife.36530
Publication status	Published - 10 Aug 2018

Keywords

Amino Acids/chemistry
Animals
Cell Line
Chromatography, High Pressure Liquid
Humans
Mice
Polyribosomes/chemistry
Protein Biosynthesis
Proteomics
Ribosomes/chemistry

ASJC Scopus subject areas

Biochemistry, Genetics and Molecular Biology(all)
Immunology and Microbiology(all)
Neuroscience(all)

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10.7554/eLife.36530Licence: CC BY

Final Published VersionFinal published version, 8.97 MBLicence: CC BY

Multidimensional Proteomic Analysis of Metabolic Stress & Cellular Phenotypes (Strategic Grant)
Cantrell, D. & Lamond, A.
Wellcome Trust
1/01/15 → 31/12/19
Project: Research

Lamond, Angus
- Gene Regulation and Expression - Professor of Biochemistry
Person: Academic

Cite this

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title = "Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC",

abstract = "We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.",

keywords = "Amino Acids/chemistry, Animals, Cell Line, Chromatography, High Pressure Liquid, Humans, Mice, Polyribosomes/chemistry, Protein Biosynthesis, Proteomics, Ribosomes/chemistry",

author = "Harunori Yoshikawa and Mark Larance and Harney, {Dylan J.} and Ramasubramanian Sundaramoorthy and Tony Ly and Thomas Owen-Hughes and Lamond, {Angus I.}",

note = "Wellcome; H2020 | H2020 Priority Excellent Science | H2020 Marie Sk{\l}odowska-Curie Actions (MSCA); Uehara Memorial Foundation; Naito Foundation; Royal Society of Edinburgh (RSE)",

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doi = "10.7554/eLife.36530",

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T1 - Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

AU - Yoshikawa, Harunori

AU - Larance, Mark

AU - Harney, Dylan J.

AU - Sundaramoorthy, Ramasubramanian

AU - Ly, Tony

AU - Owen-Hughes, Thomas

AU - Lamond, Angus I.

N1 - Wellcome; H2020 | H2020 Priority Excellent Science | H2020 Marie Skłodowska-Curie Actions (MSCA); Uehara Memorial Foundation; Naito Foundation; Royal Society of Edinburgh (RSE)

PY - 2018/8/10

Y1 - 2018/8/10

N2 - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

AB - We describe Ribo Mega-SEC, a powerful approach for the separation and biochemical analysis of mammalian polysomes and ribosomal subunits using Size Exclusion Chromatography and uHPLC. Using extracts from either cells, or tissues, polysomes can be separated within 15 min from sample injection to fraction collection. Ribo Mega-SEC shows translating ribosomes exist predominantly in polysome complexes in human cell lines and mouse liver tissue. Changes in polysomes are easily quantified between treatments, such as the cellular response to amino acid starvation. Ribo Mega-SEC is shown to provide an efficient, convenient and highly reproducible method for studying functional translation complexes. We show that Ribo Mega-SEC is readily combined with high-throughput MS-based proteomics to characterize proteins associated with polysomes and ribosomal subunits. It also facilitates isolation of complexes for electron microscopy and structural studies.

KW - Amino Acids/chemistry

KW - Animals

KW - Cell Line

KW - Chromatography, High Pressure Liquid

KW - Humans

KW - Mice

KW - Polyribosomes/chemistry

KW - Protein Biosynthesis

KW - Proteomics

KW - Ribosomes/chemistry

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DO - 10.7554/eLife.36530

M3 - Article

C2 - 30095066

SN - 2050-084X

VL - 7

SP - 1

EP - 26

JO - eLife

JF - eLife

IS - 7

M1 - e36530

ER -

Efficient Analysis of Mammalian Polysomes in cells and tissues using Ribo Mega-SEC

Abstract

Keywords

ASJC Scopus subject areas

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Projects

Multidimensional Proteomic Analysis of Metabolic Stress & Cellular Phenotypes (Strategic Grant)

Profiles

Lamond, Angus

Cite this